Field of the Invention
The present invention relates to sample observation methods and sample observation devices.
Description of the Related Art
When a sample is illuminated with parallel light flux, non-diffracted light (hereinafter, referred to as “zero-order diffracted light”) and diffracted light are generated from a sample. In microscopes, an image of a sample is formed by synthesis of zero-order diffracted light and diffracted light.
Complex amplitude E at the image plane is represented by the following expression, for example:E=A1e−iϕ1(r)eiωt+A2e−iϕ2(r)eiωt,
Where
A1 denotes an amplitude of zero-order diffracted light.
A2 denotes an amplitude of diffracted light,
ϕ1(r) denotes a phase of zero-order diffracted light, and
ϕ2(r) denotes a phase of diffracted light.
Since intensity of light is observed at the image plane, the intensity I of light at the image plane can be represented by the following expression:I=|E|2=A12+A22+2A1A2 cos ψ,
where
ψ denotes a phase difference, and ψ=ϕ1(r)−ϕ2 (r).
As described above, zero-order diffracted light and diffracted light is necessary for forming the image (optical image) of the sample. Therefore, in the following description, an image (optical image) of a sample is assumed to be formed by zero-order diffracted light and first-order diffracted light. Since the phase of first-order diffracted light delays π/2 relative to the phase of zero-order diffracted light, the phase difference is expressed by ψ=0−(−π/2)=π/2. In this case, since 2A1A2 cos ψ=0, phase information cannot be obtained in the form of contrast information. As a result, in attempting to observe an image of a colorless and transparent sample, e.g., cells, at an in-focus position, it is very difficult to observe the image of the cell in a bright-field observation.
A phase-contrast observation is one method to observe the colorless and transparent sample. In the phase-contrast observation, a phase-contrast microscope is used. Various proposals have been made for a phase-contrast microscope. A microscope, which makes an observation of the sample at a position displaced from the in-focus position of an image forming optical system so as to observe an image (phase-contrast image) in a wide observation field, is one available microscope. The microscope disclosed in Japanese Patent Application Laid-open Publication No. 2005-173288 (hereinafter, referred to as “JP 2005-173288 A”) includes a partial aperture and wavefront introduction means. The partial aperture is located substantially at a pupil position of the illumination optical system, and the wavefront introduction means is located at the pupil position of the image forming optical system. Moreover, the wavefront introduction means introduces a wavefront that varies in size with the pupil diameter of the image forming optical system.
When the sample is displaced from the in-focus position of the image forming optical system, a difference in optical path length (phase difference) occurs between zero-order diffracted light and diffracted light. In this case, since 2A1A2 cos ψ≠0, then phase information can be obtained in the form of contrast information. The value of A12, however, is very large as compared with the value of 2A1A2 cos ψ. Therefore, in the microscope of JP 2005-173288 A, the wavefront introduction means, i.e., an absorption film, is located at the pupil position of the image forming optical system, and whereby the value of A1 is reduced.